Riboflavin fortification of rice endosperm by metabolic engineering

Tian, Yong‐Sheng; Xu, Jing; Wang, Bo; Fu, Xin; Gao, Jian‐Jie; Han, Ho Jae; Li, Zhenjun; Wang, Lijuan; Zhang, Fu-Jian; Zhang, Wenhui; Deng, Yong‐Dong; Wang, Yu; Peng, Ri‐He; Yao, Quan‐Hong

doi:10.1111/pbi.13615

Cited by 8 publications

(7 citation statements)

References 11 publications

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“…In contrast to plants, fungi and bacteria, humans do not have the ability to produce riboflavin de novo and rely solely on dietary intake to meet the daily requirements [ 2 , 5 , 12 ]. The deficiency in riboflavin, known as ariboflavinosis, is a global health concern that can result in various disorders, such as cardiovascular diseases, anemia, cancer and neurological and developmental conditions [ 13 ].…”

Section: Introductionmentioning

confidence: 99%

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Mathematical kinetic modelling followed by in vitro and in vivo assays reveal the bifunctional rice GTPCHII/DHBPS enzymes and demonstrate the key roles of OsRibA proteins in the vitamin B2 pathway

Faustino,

Lourenço,

Strobbe

et al. 2024

BMC Plant Biol

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Background Riboflavin is the precursor of several cofactors essential for normal physical and cognitive development, but only plants and some microorganisms can produce it. Humans thus rely on their dietary intake, which at a global level is mainly constituted by cereals (> 50%). Understanding the riboflavin biosynthesis players is key for advancing our knowledge on this essential pathway and can hold promise for biofortification strategies in major crop species. In some bacteria and in Arabidopsis, it is known that RibA1 is a bifunctional protein with distinct GTP cyclohydrolase II (GTPCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) domains. Arabidopsis harbors three RibA isoforms, but only one retained its bifunctionality. In rice, however, the identification and characterization of RibA has not yet been described. Results Through mathematical kinetic modeling, we identified RibA as the rate-limiting step of riboflavin pathway and by bioinformatic analysis we confirmed that rice RibA proteins carry both domains, DHBPS and GTPCHII. Phylogenetic analysis revealed that OsRibA isoforms 1 and 2 are similar to Arabidopsis bifunctional RibA1. Heterologous expression of OsRibA1 completely restored the growth of the rib3∆ yeast mutant, lacking DHBPS expression, while causing a 60% growth improvement of the rib1∆ mutant, lacking GTPCHII activity. Regarding OsRibA2, its heterologous expression fully complemented GTPCHII activity, and improved rib3∆ growth by 30%. In vitro activity assays confirmed that both OsRibA1 and OsRibA2 proteins carry GTPCHII/DHBPS activities, but that OsRibA1 has higher DHBPS activity. The overexpression of OsRibA1 in rice callus resulted in a 28% increase in riboflavin content. Conclusions Our study elucidates the critical role of RibA in rice riboflavin biosynthesis pathway, establishing it as the rate-limiting step in the pathway. By identifying and characterizing OsRibA1 and OsRibA2, showcasing their GTPCHII and DHBPS activities, we have advanced the understanding of riboflavin biosynthesis in this staple crop. We further demonstrated that OsRibA1 overexpression in rice callus increases its riboflavin content, providing supporting information for bioengineering efforts.

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Section: Introductionmentioning

confidence: 99%

“…Biofortification of riboflavin in rice has been previously performed by overexpressing six out of the seven genes that constitute the yeast biosynthetic pathway [ 12 ]. However, recent trends in biofortification suggest a shift towards the overexpression of endogenous genes, reflecting public consumer preferences [ 42 , 43 ].…”

Section: Introductionmentioning

confidence: 99%

Mathematical kinetic modelling followed by in vitro and in vivo assays reveal the bifunctional rice GTPCHII/DHBPS enzymes and demonstrate the key roles of OsRibA proteins in the vitamin B2 pathway

Faustino,

Lourenço,

Strobbe

et al. 2024

BMC Plant Biol

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show abstract

“…Studies have shown that riboflavin deficiency in less developed regions can result in clinical symptoms, such as cheilitis, glossitis, angular cheilitis, and anemia, given that the average daily dietary reference intakes of riboflavin for women and men are lower than 1.2 and 1.3 mg, respectively 20,21 . A study documented that riboflavin content could be enhanced in rice endosperm through genetic engineering 22 . Meanwhile, riboflavin is recognized as a safe and novel compound for inducing plant resistance.…”

Section: Introductionmentioning

confidence: 99%

“…20,21 A study documented that riboflavin content could be enhanced in rice endosperm through genetic engineering. 22 Meanwhile, riboflavin is recognized as a safe and novel compound for inducing plant resistance. Researchers have primarily applied riboflavin through foliar spraying and soil drenching to investigate its effects.…”

Section: Introductionmentioning

confidence: 99%

Riboflavin improves grain yield, 2‐acetyl‐1‐pyrroline accumulation, and antioxidative properties of fragrant rice

Cheng,

Liu,

et al. 2023

J Sci Food Agric

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BackgroundRiboflavin, a vital water‐soluble vitamin with antioxidative activity, plays a critical role in maintaining overall bodily health and defense responses. However, its impact on fragrant rice yield and aroma remains unexplored.ResultsIn a 2022 pot experiment with Meixiangzhan and Yuxiangyouzhan fragrant rice cultivars, we applied riboflavin foliar treatments at concentrations of 0 (CK), 10 (R10), 20 (R20), and 40 (R40) mg·L‐1 during the initial heading stage. Riboflavin increased rice yield, 2‐AP content, and antioxidative properties. It boosted 2‐AP level by 13.1‐50.1% for Meixiangzhan and 22.3%‐35.3% for Yuxiangyouzhan, with the highest levels in R20 and R10 treatments. This increase is significantly correlated with elevated levels of proline, pyrroline‐5‐carboxylic acid, 1‐pyrroline, and methylglyoxal, as well as heightened enzyme activities, including those of proline dehydrogenase, ornithine aminotransferase, and pyrroline‐5‐carboxylic acid synthetase (P5CS). The R20 treatment resulted in the highest yield due to an improved seed‐setting rate. Importantly, a positive correlation emerged between 2‐AP content and yield, both significantly linked to superoxide dismutase, proline, hydrogen peroxide, P5CS, catalase, and 1‐pyrroline.ConclusionsRiboflavin maintained enzyme activities, regulated substance synthesis pathways, and increased 2‐AP and yield, especially in the R20 treatment. These insights advance fragrant rice production theory by uncovering riboflavin's role in the development of fragrant rice.This article is protected by copyright. All rights reserved.

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“…To maintain a single transgenic locus, developers can combine new transgenes with previously introduced transgenes in vitro for a new round of transformation [ 2 , 3 , 4 ]. Making a longer transformation construct may not be technically difficult, but to find all the genes appropriately expressed in a single integration event could be challenging.…”

Section: Introductionmentioning

confidence: 99%

Target Lines for in Planta Gene Stacking in Japonica Rice

Han

Yin

et al. 2022

IJMS

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The clustering of transgenes at a chromosome location minimizes the number of segregating loci that needs to be introgressed to field cultivars. Transgenes could be efficiently stacked through site-specific recombination and a recombinase-mediated in planta gene stacking process was described previously in tobacco based on the Mycobacteriophage Bxb1 site-specific integration system. Since this process requires a recombination site in the genome, this work describes the generation of target sites in the Japonica rice genome. Agrobacterium-mediated gene transfer yielded ~4000 random-insertion lines. Seven lines met the criteria of being single copy, not close to a centromere, not inserted within or close to a known gene or repetitive DNA, having precise recombination site sequences on both ends, and able to express the reporter gene. Each target line tested was able to accept the site-specific integration of a new gfp-containing plasmid and in three of those lines, we regenerated fertile plants. These target lines could be used as foundation lines for stacking new traits into Japonica rice.

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Riboflavin fortification of rice endosperm by metabolic engineering

Cited by 8 publications

References 11 publications

Mathematical kinetic modelling followed by in vitro and in vivo assays reveal the bifunctional rice GTPCHII/DHBPS enzymes and demonstrate the key roles of OsRibA proteins in the vitamin B2 pathway

Mathematical kinetic modelling followed by in vitro and in vivo assays reveal the bifunctional rice GTPCHII/DHBPS enzymes and demonstrate the key roles of OsRibA proteins in the vitamin B2 pathway

Riboflavin improves grain yield, 2‐acetyl‐1‐pyrroline accumulation, and antioxidative properties of fragrant rice

Target Lines for in Planta Gene Stacking in Japonica Rice

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